Homogenates Of Skin Fibroblasts Research Articles

Saposins ( sap) A and C activate the degradation of galactosylsphingosine

As previously shown for [ 3H-galactosyl]ceramide, the breakdown of [ 3H-galactosyl]sphingosine was reduced in prosaposin-deficient skin fibroblast homogenates. Galactosylsphingosine hydrolysis was also deficient in cell homogenates from Krabbe’s disease (β-galactocerebrosidase-deficient) patients, but not acid β-galactosidase-deficient patients. Moreover, hydrolysis of galactosylsphingosine in the prosaposin-deficient cell homogenates could be partially restored by adding pure saposin A or C, thereby identifying these saposins as essential facilitators of galactosylsphingosine hydrolysis. By contrast, saposins B and D had little effect on galactosylsphingosine hydrolysis in the prosaposin-deficient cells. The reduced galactosylsphingosine turnover in prosaposin-deficiency suggests that there could be a pathogenetic cerebral accumulation of galactosylsphingosine in this disorder.

Quantification of Cellular Acid Sphingomyelinase and Galactocerebroside β-Galactosidase Activities by Electrospray Ionization Mass Spectrometry

Diagnosis of Niemann-Pick (A and B) and Krabbe diseases is achieved by measurement of the lysosomal enzymes acid sphingomyelinase (ASM) and galactocerebroside beta-galactosidase (GCG), respectively. Conventional assays use radiolabeled or fluorescent substrates and do not allow simultaneous determination of two or more enzymes in the sample. We developed a sensitive and specific method to assay ASM and GCG in skin fibroblast homogenates using biotinylated substrate conjugates. The products were purified by bioaffinity capture on streptavidin-agarose beads and, following release, were analyzed by electrospray ionization mass spectrometry. Quantification was achieved using stable-isotope-labeled internal standards that were chemically identical to the products of the enzymatic reactions. The method demonstrated excellent linearity of ASM and GCG enzymatic product formation with the amount of cellular protein and incubation time. The range of ASM activities in fibroblast lysates from six healthy patients was 39-70 nmol. mg(-1). h(-1) compared with 3.7-5.1 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Niemann-Pick A disease. The GCG activities toward the corresponding substrate conjugate were 4.0-6.8 nmol. mg(-1). h(-1) in cell lysates from healthy patients compared with 0.1-0.2 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Krabbe disease. The amounts of substrate conjugates needed per analysis were 15 nmol (14 microg) for both ASM and GCG. Electrospray mass spectrometry combined with the use of biotinylated substrate conjugates and bioaffinity purification represents a new approach for the diagnosis of lysosomal storage diseases as demonstrated for Niemann-Pick A and Krabbe diseases. No radioactive substrates are used, and the method uses a single instrumental platform to determine both ASM and GCG in one cell sample.

Relationship between monoamine oxidase (MAO) A specific activity and proportion of human skin fibroblasts which express the enzyme in culture.

Total deficiency of monoamine oxidase A (MAO-A) in affected males of a single, human kindred appears to be associated with mild mental retardation and significant behavioral anomalies. Though total MAO-A deficiency appears to be rare, the extent and significance of individual variation in monoamine oxidase A activity in human populations is unclear. Since MAO-A activity is undetectable in blood cells, most systematic surveys of individual variation MAO-A activity have compared enzyme activity in human fibroblasts cultured from skin biopsies. Surprisingly, MAO-A activity in skin fibroblast cultures from unrelated donors ranges over 100-fold. It has been suggested that this extreme variation in fibroblast MAO-A activity between donors reflects individual, genetic variation in the regulation of MAO-A in fibroblasts. I have found from studies with immunofluorescence microscopy and flow cytometry that the proportion of MAO-A+ cells in fibroblast cultures is (a) highly variable between cultures, (b) a reproducible characteristic of each culture and (c) the primary factor responsible for variation in MAO-A specific activity in whole cell, skin fibroblast homogenates. It has been shown previously that MAO-A activity of a skin fibroblast culture is relatively constant with continued passage prior to cellular senescence. Therefore, these new data raise the possibility that MAO-A expression is confined to a functionally distinct subset of human skin fibroblasts.

Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids.

Isoflavonoids and lignans, constituents of many plant foods, have been proposed as protective agents in those populations with a low incidence of hormone-dependent cancers. They may act by their inhibition of the metabolism of growth-promoting steroid hormones. This report describes the inhibition of 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase by six isoflavonoids and two lignans in human genital skin fibroblast monolayers and homogenates, and in benign prostatic hyperplasia tissue homogenates. In genital skin fibroblasts, genistein, biochanin A and equol were the most potent inhibitors of 5 alpha-reductase activity, each resulting in greater than 80% inhibition at a concentration of 100 microM. The IC50 values for genistein and a seven-compound mixture were approximately 35 microM and 20 microM (2.9 microM of each compound) respectively. Of the lignans, enterolactone was the most potent inhibitor. Inhibition by biochanin A was shown to be reversible. When genital skin fibroblast homogenates were used, biochanin A was found to inhibit 5 alpha-reductase isozymes 1 and 2 to differing extents (30% and 75% respectively). Genistein was shown to inhibit 5 alpha-reductase 2 in a non-competitive nature (Vmax and Km values without and with inhibitor were 30 and 20 pmol/mg protein per h and 177 and 170 nM respectively). All of the compounds tested inhibited 17 beta-hydroxysteroid dehydrogenase activity in genital skin fibroblast monolayers. When prostate tissue homogenates were used, the compounds tested were better inhibitors of 5 alpha-reductase 1 than 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Human glucosamine-6-sulphatase deficiency. Diagnostic enzymology towards heparin-derived trisaccharide substrates

Glucosamine-6-sulphatase (6S) activity towards a series of radiolabelled heparin-derived trisaccharide substrates was determined in cultured human skin fibroblast and leucocyte homogenates, and in urine supernatants of normal individuals and patients affected with 6S deficiency [Sanfilippo D syndrome; mucopolysaccharidosis (MPS) type IIID]. The N-sulphated and N-acetylated derivatives of the trisaccharide substrate O-(alpha-glucosamine 6-sulphate)-(1----4)-L-O-(alpha-iduronic acid 2-sulphate)-(1----4)-D-O-2,5-anhydro[1-3H]mannitol 6-sulphate (GlcNH6S-IdoA2S-anM6S) were prepared by enzymic digestion of a pentasulphated tetrasaccharide isolated following the HNO2 deamination of heparin. Purified lysosomal enzymes and MPS-patient skin fibroblasts were used along with chemical degradation to confirm the structure of each of the substrates that were utilized to study the interaction of the enzyme activities required to degrade the highly sulphated regions of heparan sulphate. Human liver, skin fibroblast and urine 6S activities were separated by chromatofocusing into at least four and possibly up to six individual activities. 6S activities present in each of the tissues generally had similar catalytic properties, including Km values, pH optima and inhibition with NaCl, Na2SO4 and NaH2PO4. Leucocyte and skin fibroblast 6S activities towards GlcNAc6S-IdoA2S-anM6S were maximal at pH 4.1 and 3.9 respectively, with Km values of 2.8 microM and 0.9-1.7 microM respectively. Urine 6S activity towards GlcNAc6S-IdoA2S-anM6S was stimulated 30-fold by BSA at pH 3.9, which shifted the pH optimum from 5.1 to 4.2 and decreased the Km value at pH 4.2 from 4.0 microM to 0.5 microM. Residual 6S activity present in the skin fibroblast homogenates from MPS IIID patients was characterized for activity towards GlcNAc6S-IdoA2S-anM6S and observed to have similar pH optima and Km values to normal skin fibroblast 6S activities, although the residual 6S activity was less than 1% of the normal control range.

Glucuronate-2-sulphatase activity in cultured human skin fibroblast homogenates

The optimization of the assay conditions to detect glucuronate-2-sulphatase (GS) activity present in cultured human skin fibroblast homogenates towards a heparin-derived disaccharide substrate O-(beta-D-glucuronic acid 2-sulphate)-(1----4)-D-O-2,5-anhydro[l-3H]mannitol 6-sulphate (GSMS) has shown that a complex relationship exists between pH, buffer composition, ionic strength and the influence of added BSA and salts (NaCl, Na2SO4, CuCl2 and ZnCl2) to achieve maximum sulphatase activity. Whereas albumin stimulated GS activity by more than 2-fold over the pH range 2.7-5.7, CuCl2 stimulated GS activity over the narrow pH range 3.0-4.2, and inhibited GS activity at higher pH. ZnCl2 stimulated GS activity more than 3-fold at pH 3.0 and by more than 10-fold at pH 4.8. NaCl inhibited GS activity at pH 3.0, while activity between pH 4.2 and 4.8 was stimulated by up to 10-fold, resulting in a shift in the observed pH optimum from 3.0 to 4.8 in the presence of 315 mM-NaCl. Skin fibroblast GS activity toward GSMS had apparent Km values of 0.5-1.2 microM at pH 3.0, and 27.0-33.2 microM at pH 4.8. Albumin stimulated GS activity at both low and high pH by an increase in the apparent Vmax. values without significant alteration in the respective Km values. At pH 4.8, NaCl stimulated GS activity as a result of an increase in Vmax. values. These observations raise the possibility that two forms of GS activity are present in skin fibroblast homogenates: a low-Km form that has a pH optimum of 3.0 and is stimulated by BSA and a high-Km form with a pH optimum of 4.8 which is stimulated by NaCl.

Pyruvate Carboxylase Deficiency: A Benign Variant with Normal Development

The devastating nature of a pyruvate carboxylase deficiency is underscored by the uniformly fatal outcome of the neonatal (French) type and the severely disabling and ultimately fatal outcome of the infantile (North American) type. We report a 7-y-old girl with metabolic and biochemical features of the North American phenotype. Remarkably, the clinical course has been benign with preservations of motor and mental abilities. The residual enzyme activity in cultured skin fibroblast homogenates was 1.8% and cross-reacting material was present in normal abundance and electrophoretic mobility. She has had several episodes of metabolic acidosis with elevated lactate, pyruvate, alanine, beta-hydroxybutyrate, acetoacetate, lysine, and proline values, and undetectably low aspartate concentrations. These crises have been managed by rehydration and bicarbonate therapy. We are unable to provide a satisfactory explanation for the uniquely benign clinical course that has been experienced by this patient.

Uptake and degradation of several pyrenesphingomyelins by skin fibroblasts from control subjects and patients with Niemann-Pick disease. Effect of the structure of the fluorescent fatty acyl residue

Three fluorescent analogues of sphingomyelin (SPM), each containing pyrene in the fatty acyl residue, were synthesized and employed for the study of their mode of uptake by, and degradation within, intact cultured human skin fibroblasts. These were prepared by condensing sphingosylphosphocholine and the following fatty acids: pyrenedodecanoic acid (P12), pyrenesulphonylaminoundecanoic acid (PSA11) and pyrenepropenoic acid (P3:1). The cell association and catabolism of these SPM analogues by normal, Niemann-Pick-disease-Type-A and low-density-lipoprotein (LDL)-receptor-negative familial hypercholesterolaemia fibroblasts were investigated and compared with the metabolism of [cholinemethyl-14C]sphingomyelin. The catabolism of the fluorescent derivatives was monitored by measuring the appearance of the corresponding fluorescent ceramides. Two modes of uptake and degradation patterns were observed. Thus P12-SPM and radiolabelled SPM were taken up by LDL-receptor-mediated endocytosis when incubated with serum-containing medium, this conclusion being supported by the very low uptake by familial-hypercholesterolaemia fibroblasts, which lack the apolipoprotein-B/E receptor. After uptake, these compounds were metabolically degraded solely by the lysosomal sphingomyelinase, as evidenced by the fact that more than 98% of the SPM remained undegraded in Niemann-Pick-disease cells. By contrast, PSA11- and P3:1-SPMs were taken up by a receptor-independent endocytic pathway, as indicated by the similar rates of uptake in control and familial-hypercholesterolaemia cells in the absence or presence of fetal-calf serum in the culture medium. The degradation of PSA11-SPM and P3:1-SPM was brought about, in the main, by the lysosomal sphingomyelinase, but also by a yet uncharacterized process. The latter catabolic pathway, active in Niemann-Pick-disease-Type-A fibroblasts, seems to differ from the neutral Mg2(+)-dependent sphingomyelinase whose activity was undetectable in homogenates of skin fibroblasts. The present study emphasizes the influence of the structure of the fatty acyl moiety of SPM on its association with lipoproteins and/or cell membranes and on its intracellular routing and metabolic degradation.

Dihydroxyacetone phosphate acyltransferase and alkyldihydroxyacetone phosphate synthase activities in rat liver subcellular fractions and human skin fibroblasts

Dihydroxyacetone phosphate acyltransferase (DHAP-AT) and alkyldihydroxyacetone phosphate synthase (DHAP-synthase) activities were examined in subcellular fractions of rat liver. The results indicate that at least 80% of DHAP-AT (assays carried out at pH 5.4) activity in rat liver is in peroxisomes, and the remaining activity is mitochondrial. In contrast to DHAP-AT, DHAP-synthase was detected in all subcellular fractions analyzed but the activity in peroxisomes was 208-fold and 42-fold greater compared to mitochondria and microsomes, respectively. We estimate that at least 70% of the DHAP-synthase activity in rat liver is in peroxisomes. DHAP-AT and DHAP-synthase activities were also examined in homogenates of skin fibroblasts from patients with inherited defects in peroxisomal structure and/or function. Both the enzyme activities were deficient in Zellweger syndrome whereas the activities were only partially deficient in infantile Refsum's disease. Greater reduction in DHAP-synthase activity, but only a partial reduction in DHAP-AT activity was observed in rhizomelic chondrodysplasia punctata. However, both DHAP-AT and DHAP-synthase activities were either normal or near normal in Refsum's disease or X-linked adrenoleukodystrophy. The results reported suggest that various peroxisomal disease states can be identified based on DHAP-AT and DHAP-synthase activities in skin fibroblasts of patients.

β-oxidation of very-long-chain fatty acids and their coenzyme A derivatives by human skin fibroblasts

The β-oxidation of lignoceric acid (C24:0), hexacosanoic acid (C26:0), and their coenzyme A derivatives was investigated in human skin fibroblast homogenates. The cofactor requirements for oxidation of lignoceric acid and hexacosanoic acid were identical but were different from their coenzyme A derivatives. For example, lignoceric acid and hexacosanoic acid oxidation was strictly ATP dependent whereas the oxidation of the corresponding coenzyme A derivatives was ATP independent. Also the rate of oxidation of coenzyme A derivatives of lignoceric acid or hexacosanoic acid was much higher compared to the free fatty acids. In patients with Zellweger's syndrome, X-linked adrenoleukodystrophy and infantile Refsum's disease, the β-oxidation of lignoceric and hexacosanoic acids was defective whereas the oxidation of their corresponding coenzyme A derivatives was nearly normal. The results presented in this communication suggest strongly that the β-oxidation of very-long-chain fatty acids occurs exclusively in peroxisomes. However, the coenzyme A derivatives of very-long-chain fatty acids can be oxidized in mitochondria as well as in peroxisomes. The inability of the mitochondrial system to oxidize free fatty acids may be due to its inability to convert them to their corresponding coenzyme A derivatives. Our results suggest that a specific very-longchain fatty acyl CoA synthetase may be required for the activation of the free fatty acids and that this synthetase may be deficient in patients with Zellweger's syndrome and possibly X-linked adrenoleukodystrophy, as well. The results presented suggest that substrate specificity and the subcellular localization of the synthetase may regulate the β-oxidation of very-long-chain fatty acids in the cell.

A comparative study of stearic and lignoceric acid oxidation by human skin fibroblasts

Sensitive assays were developed for long chain and very long chain fatty acid oxidation in human skin fibroblast homogenates. Stearic and lignoceric acids were degraded by the fibroblasts by the β-oxidation pathway. The cofactor requirements for stearic and lignoceric acid β-oxidation were very similar but not identical. For example, appreciable lignoceric acid oxidation could be demonstrated only in the presence of α-cyclodextrin and was inhibited by Triton X-100. In Zellweger's syndrome, stearic acid β-oxidation was partially reduced whereas lignoceric acid β-oxidation was reduced dramatically (< 12% activity compared to the controls). The results presented suggest that stearic acid β-oxidation occurs in mitochondria as well as in peroxisomes, but lignoceric acid oxidation occurs entirely in the peroxisomes. We suggest that the β-oxidation systems for stearic acid and lignoceric acid may be different.

A microassay for acid β-galactosidase activity toward asialofetuin

To study the enzymatic properties of β-galactosidase from the patients with a β-galactosidase deficiency such as G M1 gangliosidosis, determination of enzymatic activity with naturally occurring substrates, asialofetuin in addition to another natural substrate, G M1 ganglioside, is essentially required. With a previously reported, simple and sensitive fluorometric assay for G M1 ganglioside β-galactosidase using high performance liquid chromatography (HPLC), optimal reaction conditions were determined for the assay of acid β-galactosidase activity toward asialofetuin in skin fibroblast homogenates. Under these conditions, reduced enzymatic activities could be detected in cultured skin fibroblasts from patients with type 1 and 3 G M1 gangliosidoses and mucopolysaccharidosis IV-B (Morquio B syndrome). This method was applicable to study of the enzymatic properties of the mutant β-galactosidase and provided an alternative to assays employing radioactive or artificial substrates.

Studies on the activation of sphingomyelinase activity in Niemann-Pick type A, B, and C fibroblasts: enzymological differentiation of types A and B.

Cultured skin fibroblast homogenates from patients with Niemann-Pick disease Type C, were able to degrade sphingomyelin liposomes at a normal rate. Fibroblasts from patients with Niemann-Pick disease Types A and B were less active (0.08-0.55 versus 0.96-2.93 nmol/h/mg). When fibroblasts were maintained in synthetic media (MCDB-104) devoid of fetal calf serum for a period of 21 days, sphingomyelinase activity measured at pH 3.8 increased in control and Niemann-Pick Type C (up to 15-fold) and in Niemann-Pick Type B (up to 3-fold) while Niemann-Pick Type A showed no significant increase in sphingomyelinase activity. Addition of a protein activator isolated from the spleen of a Type I Gaucher's disease patient stimulated a 2-7.5-fold increase in sphingomyelinase activity in normal, Niemann-Pick Type B and C fibroblasts, while under the same conditions the Niemann-Pick Type A fibroblast enzyme responded poorly. Our data show that the residual sphingomyelinase activity in Niemann-Pick Type A can be differentiated from that present in other phenotypic forms by its lack of response to the Gaucher activator. Furthermore, we can find no evidence to support the view that Niemann-Pick Type C sphingomyelinase differs from the normal enzyme in its response to Gaucher activator.

Application of a G M1 ganglioside β-galactosidase microassay method to diagnosis of G M1 gangliosidosis

The enzymatic diagnosis of G M1 gangliosidosis, including the diagnosis of heterozygosity, requires a microassay of G M1 ganglioside β-galactosidase activity in lymphocytes and cultured skin fibroblasts. We have adopted high-performance liquid chromatography (HPLC) to the assay of this enzyme and can measure the activity in crude samples fluorometrically. Reaction conditions were examined to determine those optimal for the assay of G M1 ganglioside β-galactosidase activity in lymphocyte and skin fibroblast homogenates. Under these optimal conditions, reduced enzymatic activities could be detected in lymphocytes and cultured skin fibroblasts from three patients with G M1 gangliosidosis. Thus, this assay can be used for the diagnosis, rather than the usual assays employing radioactive or artificial substrates.

A fluorometric microassay procedure for monitoring the enzymatic activity of GM 1-ganglioside β-galactosidase by use of high-performance liquid chromatography

For the measurement of the enzymatic activity of GM 1-ganglioside (II 3 NeuAcGgOse 4Cer, galactosyl- N-acetylgalactosaminyl-( N-acetylneuraminosyl)-galactosyl-glucosylceramide) β-galactosidase in crude enzyme samples, a microassay using nonradioisotopic GM 1-ganglioside was devised. To reduce the volume of the reaction mixture and eliminate the interferences due to the fluorescent contaminants in the reaction mixture, NADH, a product after the oxidation of the released galactose with NAD and β-galactose dehydrogenase, was fluorometrically estimated by use of high-performance liquid chromatography. By this method, as little as 10 pmol of galactose can be detected. Using rat brain homogenates as an enzyme sample, the several parameters were reexamined to define the optimal conditions for the assay. This assay method was also applied to human cultured skin fibroblast homogenates, and it was found that this method can be used for the diagnosis of GM 1-gangliosidosis, instead of the usual method using the radioisotopelabeled natural substrate.

Alkaline phosphatase deficiency in cultured skin fibroblasts from patients with hypophosphatasia: comparison of the infantile, childhood, and adult forms.

Alkaline phosphatase (AP) activity was assayed by a sensitive fluorometric method, using 4-methylumbelliferyl phosphate as substrate, in homogenates of confluent skin fibroblasts from 17 patients with the infantile, childhood, or adult form of hypophosphatasia and compared to the AP activity in 22 sex- and age-matched control cell lines. Statistically significant deficiency of AP activity was found in all 3 types of hypophosphatasia (8.2%, 30.2% and 18.5% of control values, respectively); however, there was considerable variation of enzyme activity, especially in the control groups, and some overlap between patient and control values occurred for the childhood and adult age groups. These fibroblast studies suggest that all clinical forms of hypophosphatasia are characterized by deficient activity of the bone/liver/kidney AP isoenzyme. The clinical severity of the infantile form is reflected by a profound lack of AP activity in cultured skin fibroblasts. Further study will be necessary to determine the mechanism(s) of the deficiency in AP activity in each form of hypophosphatasia. Nevertheless, we suggest that AP-deficient infantile hypophosphatasia skin fibroblasts in culture might constitute a good tool by which to explore the unknown physiological role of AP.

Four sanfilippo C patients in a family pedigree

AbstractThe deficiency of acetyl‐CoA:α‐glucosaminideN‐acetyltransferase was detected in skin fibroblast homogenates from four patients in a family pedigree. Further, it was observed that the relative proportion of the acetyl groups in heparan sulphate from a Sanfilippo C patient was much lower than those obtained from Sanfilippo A and B patients.

Deficient phosphorylation of mannose residues of mannan in fibroblasts of patients with mucolipidoses II and III.

Incorporation of 32P from [gamma 32P]ATP into mannan could not be detected in homogenates of cultivated skin fibroblasts from patients with mucolipidosis II, and accounted for only up to 10% of normal control activity in cell lysates from patients with mucolipidosis III. Parents of patients with mucolipidosis II demonstrated 60-70% of normal control activity. On high-voltage electrophoresis, the hydrolysed mannan from reactions performed with normal cells, over the pH range 5.5-7.5, yielded a radioactive band migrating with the same mobility as mannose 6-phosphate, whereas no such product could be demonstrated in fibroblasts of patients with mucolipidosis II.

Isolation and use of a new natural substrate for the diagnosis of MPS VII

Mucopolysaccharidosis Type VII is characterized by a severe deficiency of the enzyme β-D-glucuronidase. As with other mucopolysaccharidoses, this inherited enzyme disorder results in a wide range of phenotypes. from extremely mild to severe. Diagnosis of the enzyme deficiency has usually been made using artificial substrates such as 4-methylumbelliferyl-β-D-glucuronide (MuGlcUA), and p-nitrophenyl-β-D-glucuronide. We now report the isolation of a tritium-labelled disaccharide, namely glucuronosyl anhydromannitol 6-sulphate (GMs) derived from the controlled degradation of heparin. Under optimal assay conditions for normal skin fibroblast homogenates, this natural substrate gives β-D-glucuronidase activities of 0-2.4, 9 17 and 38 56 pmol/min/mg protein for homozygote affected, heterozygote and normal cell lines, respectively. Compared with the artificial substrate MuGlcUA, normal activity measured with GMs demonstrates a similar pH profile and time course, but a changed response to protein levels, greater heat sensitivity, and also lower Km and Vmax values. GMs has a similar structure to iduronosyl anhydromannitol 6-sulphate (IMs), a natural substrate of α-L-iduronidase. However, in contrast to α-L-iduronidase, β-D-glucuromdase is inhibited only slightly by SO 4 2 or Cu2+ ions; while NaCl is a much more effective inhibitor at lower pH values. This suggests that β-D-glucuronidase may-lack the specific SO 4 2 and Cu2+ binding sites postulated for α-L-iduronidase. Mucopolysaccharidosis Type VII is characterized by a severe deficiency of the enzyme β-D-glucuronidase. As with other mucopolysaccharidoses, this inherited enzyme disorder results in a wide range of phenotypes. from extremely mild to severe. Diagnosis of the enzyme deficiency has usually been made using artificial substrates such as 4-methylumbelliferyl-β-D-glucuronide (MuGlcUA), and p-nitrophenyl-β-D-glucuronide. We now report the isolation of a tritium-labelled disaccharide, namely glucuronosyl anhydromannitol 6-sulphate (GMs) derived from the controlled degradation of heparin. Under optimal assay conditions for normal skin fibroblast homogenates, this natural substrate gives β-D-glucuronidase activities of 0-2.4, 9 17 and 38 56 pmol/min/mg protein for homozygote affected, heterozygote and normal cell lines, respectively. Compared with the artificial substrate MuGlcUA, normal activity measured with GMs demonstrates a similar pH profile and time course, but a changed response to protein levels, greater heat sensitivity, and also lower Km and Vmax values. GMs has a similar structure to iduronosyl anhydromannitol 6-sulphate (IMs), a natural substrate of α-L-iduronidase. However, in contrast to α-L-iduronidase, β-D-glucuromdase is inhibited only slightly by SO 4 2 or Cu2+ ions; while NaCl is a much more effective inhibitor at lower pH values. This suggests that β-D-glucuronidase may-lack the specific SO 4 2 and Cu2+ binding sites postulated for α-L-iduronidase.

Characterization of dermatan sulfate in mucopolysaccharidosis VI. Evidence for the absence of hyaluronidase-like enzymes in human skin fibroblasts.

Dermatan sulfate-chondroitin sulfate copolymers with a high content of dermatan sulfate are stored in cultured human skin fibroblasts from patients affected with mucopolysaccharidosis VI (Maroteaux-Lamy disease). Characterization of the storage material provided evidence that hyaluronidase-like enzymes are not present in these fibroblasts. This is based on the following observations: (i) dermatan sulfate chains stored intracellularly show no reduction of molecular size as compared with intact chains isolated from the extra-cellular space; (ii) the stored dermatan sulfate chains lack reducible end groups generated by endoglycosidases; (iii) homogenates of human skin fibroblasts do not degrade hyaluronate and (iv) the stored dermatan sulfate chains are degraded by testes hyaluronidase.